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Phosphorylating Oligos

Molecular Cloning Forum

DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.



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Old 02-20-2008, 01:28 AM
Pipette Filler
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Default Phosphorylating Oligos

Heya dudes, i am in the process of phosphorylating some oligos using the enzyme PNK.

I added the following:
  • 5ul of DNA (10pM)
  • 1ul ATP (100mM)
  • 1ul 10x buffer
  • 1ul PNK
  • 2ul H2O
Samples were incubated at 37°C for 2h

However it seems my Phosphorylation protocol failed.


All of cloning were were negative! . What should i do?

My ideas it may be the ATP gone old/bad?

I was using ATP (100mM) from which was prepared as a solution from Roche.

The new ATP comes as a powder, and I make 100mM solution by dissolving it in water?

is this the problem?

thnx in advance
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oligonucleotides, oligos, phosphorylation, pnk

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